APPLICANT'S ABSTRACT: This work focuses on electrophoretic variants of arylsulfatase A (ASA; E.C. 3.1.6.1.) which have been proposed as a predisposing factor for the neuropathological effects of alcohol. This enzyme catalyzes the degradation of sulfatides to cerebrosides and sulfate. The studies in this grant application will examine genetic, metabolic, and structural aspects related to the proposed defect in alcoholic patients. To investigate the possibility that variant ASA's may be under genetic control, blood samples will be taken from first and second degree relatives of probands with variant ASA's. The platelet lysates will be analyzed for ASA banding patterns by discontinuous electrophoresis, isoelectric focusing, and two-dimensional gel electrophoresis. The possible metabolic consequences of the variant ASA's will be examined, first, by measuring the urinary levels of sulfatides in subjects with variant and normal ASA's and, second, by determining the kinetic parameters of the variant and normal ASA's in vitro using sulfatides as substrate in the presence of activator proteins. Extensive structural studies will be performed which include glycan, protein, and nucleic acid analyses. The effect of specific hydrolytic enzymes and lectin binding on variant and normal ASA's will be studied to explore the importance of structural elements of the glycan moiety on the electrophoretic pattern. The sugar content of the native enzyme as well as selected polypeptides will be determined after alkylation and fragmentation of the variant and normal ASA's. Complete structure of the glycan moiety of selected glycopeptides will be determined. Similarly, protein analysis, including comparisons of the variant forms by peptide mapping and microsequencing, will be completed for the native and deglycosylated ASA's. It will be determined if the 2.0 and 3.5 kb mRNA's for ASA arise from a single gene or two closely homologous genes. If the results from the peptide and carbohydrate analyses suggest that the variant forms of the enzyme have a different peptide backbone, the cDNA's will be used as probes to the DNA from patients with variant ASA's in order to determine if a mutation has occurred in their genome.